A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance

SUMMARY Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.


In brief
A histone H3-H4 binding domain in the Mrc1/CLASPIN component of the replisome is required for parental histone transfer and epigenetic inheritance of heterochromatin.The predicted location of Mrc1 and other histone-binding proteins in the replisome suggests a path for parental histone transfer to newly synthesized DNA.

INTRODUCTION
2][3][4][5][6] Recent studies in the fission yeast Schizosaccharomyces pombe have demonstrated that histone H3 lysine 9 trimethylation (H3K9me3), which mediates heterochromatin formation, can be epigenetically inherited independently of DNA sequence. 7,8][9][10] Following DNA replication, this read and write mechanism is thought to copy the methylation on parentally inherited histones onto newly deposited ones to restore H3K9me3 domains and gene silencing.A corollary of this model is that parental histones must be maintained during DNA replication so that the epigenetic information they contain is copied following DNA replication.1][22][23] In addition, histone-binding proteins that are either replisome components or replisome-associated histone chaperones have been identified. 4,519,24 However, how nucleosomal histones are moved across long distances from the front of the replication fork to the newly synthesized DNA at the back of the fork is not understood.
The nucleosome is composed of 147 base pairs of DNA wrapped around an octameric histone complex containing two H2A-H2B dimers and a core H3-H4 tetramer. 25,26During DNA replication, H3 and H4 are transferred as an intact tetramer. 27lthough H2A-H2B are more dynamic, recent evidence suggests that some modified H2A-H2B are also recycled during DNA replication. 19,28Multiple replisome components have been shown to bind histone H3-H4, including the Mcm2 subunit of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase, 14,15,[29][30][31][32][33][34] the Pol1 catalytic subunit of the DNA polymerase a, 19,24,35 the Dpb3-Dpb4 subunits of DNA polymerase ε, 16,36 the single-strand binding protein complex RPA, 37 and the replication licensing factor Mcm10. 38 Examination of histone association with each newly synthesized DNA strand indicates that distinct replisome components are required for symmetrical distribution of parental histones to the leading and lagging DNA strands. 15,16,39,40Mutations in the Mcm2 or Pol1 histone-binding domains (HBDs), or mutations that disrupt the coupling of the CMG helicase and DNA polymerase a via Ctf4, lead to preferential histone transfer to the leading strand, 14,15,19,24 whereas deletion of genes encoding Dpb3 or Dpb4 results in biased histone transfer to the lagging strand. 16Although parental H3-H4 are transferred as intact tetramers, 27 no replisome component that can bind to and stabilize H3-H4 tetramers has yet been identified.In addition to the above proteins, the facilitates chromatin transactions (FACT) complex, which has histone H2A-H2B and H3-H4 chaperone activities [41][42][43][44][45][46][47][48] and can mediate nucleosome retention during transcription elongation, 49,50 is associated with the replisome. 51FACT is required for efficient replication through chromatin in vitro 52 and promotes replication-coupled nucleosome assembly, 53 but whether FACT also plays a role in replication-coupled histone transfer remains unknown.
We previously used a system for inducible establishment of an ectopic domain of heterochromatin in the fission yeast S. pombe to study epigenetic inheritance (Figure 1A). 7,8In this system, a 10XtetO-ade6 + reporter gene is inserted at a euchromatic locus and recruits an engineered protein in which the bacterial tetracycline repressor (TetR) is fused to the catalytic domain of H3K9 methyltransferase Clr4 (TetR-Clr4DCD or TetR-Clr4-initiator, TetR-Clr4-I).The recruitment of TetR-Clr4-I to the tetO array results in the formation of $45 kb H3K9me2/3 domain and silencing of the ade6 + reporter gene, leading to the formation of red colonies. 7Growth in the presence of anhydrotetracycline (AHT), which releases TetR-Clr4-I from the 10XtetO sequence, then allows epigenetic inheritance of heterochromatin to be uncoupled from its sequence-dependent establishment.The separation of heterochromatin establishment and maintenance is particularly useful for uncovering mutations that disrupt parental histone transfer as such mutations would be expected to be specifically defective in heterochromatin maintenance.Using this system, a genome-wide mutagenesis screen identified mutations in several pathways that are specifically required for heterochromatin maintenance, including known heterochromatinassociated factors and the replisome. 21n this study, we report on the role of the replisome and its associated histone chaperones in heterochromatin maintenance.Using the inducible heterochromatin system, in combina-tion with biochemical, in vivo, and structural prediction approaches, we identify a role for a conserved component of the replication fork protection complex (FPC), Mrc1/CLASPIN, as a histone H3-H4 tetramer chaperone required for heterochromatin maintenance and efficient recycling of parental histones during DNA replication.We further identify FACT binding sites in the replisome, including Swi1, another subunit of the FPC and Pol1, with essential roles in heterochromatin maintenance.AlphaFold-Multimer-guided structural predictions suggest the locations of the histone-binding domains of Mrc1 and the FACT complex relative to other histone-binding proteins on the replisome.Our findings suggest a model for the transfer of parental H3-H4 tetramers to the newly synthesized leading and lagging DNA strands from an Mrc1 distribution center at the leading edge of the replication fork.

Replisome components and histone chaperones required for heterochromatin maintenance
Previous studies have shown that mutations in several replisome components, including Mrc1 and subunits with histone-binding activity, have defects in gene silencing 14,16,21,32,35,54 (Figures S1A and S1B).However, whether these mutations cause defects in the establishment and/or maintenance of silencing has been unclear.We introduced nonsense mutations in mrc1 (mrc1-W620*), 21 or mutations in genes encoding histone-binding proteins, mcm2-3A, 32 pol1-6A, 35 dpb3D, and dpb4D in cells carrying the 10XtetO-ade6 + reporter, and found that maintenance of heterochromatin was defective in cells carrying each of the above mutations, suggesting a requirement for Mcm2, Pol1, and Dpb3-Dpb4 in heterochromatin maintenance (Figure S1B).Maintenance of heterochromatin did not require the non-essential RPA subunit Rfa3 or the alternative clamp loader subunit Ctf18 (Figure S1B), the absence of which was previously shown to have severe synthetic growth defects in combination with mcm2-3A in Saccharomyces cerevisiae. 32Consistent with derepression of the ade6 + reporter gene, chromatin immunoprecipitation (ChIP)-qPCR experiments showed that in contrast to mcm2 + cells, H3K9me2 was not maintained in mcm2-3A cells 24 h after the release of the TetR-Clr4-I by growth in AHT-containing medium (Figures S1C and S1D).(C) Heterochromatin maintenance assay testing the roles of subunits of the fork protection complex in epigenetic inheritance.10-fold serial dilutions of cells were plated on the indicated growth medium to detect heterochromatin establishment (ÀAHT) and maintenance (+AHT).Loss of growth on medium containing hydroxyurea (HU+) indicates deficiency in replication checkpoint.* denotes a stop codon.(D) H3K9me2 ChIP-qPCR at the 10XtetO-ade6 + locus showing that the H3K9me2 levels in mrc1 + or mrc1D cells at the establishment phase (ÀAHT) and the maintenance phase 24 h after growth in the presence of AHT.Error bars indicate standard deviations of 3 biological replicates.(E) Diagram illustrating the gene-targeted random mutagenesis of mrc1 + to isolate mutant cells that are competent for heterochromatin establishment and replication checkpoint but fail to maintain heterochromatin.(F) Separation-of-function alleles isolated from the random mutagenesis of mrc1 + that abolishes heterochromatin maintenance but not replication checkpoint.(G) IP-MS analysis of TAP-tagged heterochromatin maintenance-competent Mrc1-SSAA and mutant Mrc1-(1-620).(H) IP-MS of TAP-Sld5 in mrc1 + and mrc1-W620STOP cells.(G and H) x axis, the log 2 fold change between wild type and mutant epitope tagged proteins; y axis, normalized intensity of proteins associated with the indicated tagged proteins detected by mass spectrometry.See also Figure S1.

Separable roles of Mrc1 in replication checkpoint signaling and epigenetic inheritance
][73] Together with two other replication proteins, Swi1/TIMELESS and Swi3/TIPIN, Mrc1/CLASPIN forms the FPC [74][75][76] (Figure 1B).We found that like Mrc1, Swi1 and Swi3 were required for the maintenance of heterochromatin (Figure 1C), indicating that the full FPC was required for heterochromatin maintenance.Consistent with its heterochromatin maintenance defects, the H3K9me2 domain at the ectopic locus was not maintained in mrc1D cells (Figure 1D).
We next tested whether the replication checkpoint function of Mrc1 in resolving replication stress was required for heterochromatin maintenance.8][79] In S. pombe, two redundant hyperphosphorylated TQs motifs (T645, T653) and one supportive SQ motif (S604) have been identified as the recruitment sites for Cds1 (Figure S1G). 79The mrc1-W620* mutation produces a truncated protein that lacks the former SQ/TQ motifs 21 (Figure S1G).We introduced mrc1-T645A, mrc1-T653A, and mrc1-S604A single and mrc1-T645A,T653A (mrc1-T2A) double amino acid substitutions into cells carrying the 10XtetO-ade6 + reporter.Cells carrying the mrc1-T2A mutations became sensitive to hydroxyurea (HU), similar to mrc1D cells, indicating sensitivity to replication stress but were competent in heterochromatin maintenance (Figure S1I).Consistently, heterochromatin maintenance did not require the checkpoint effector Cds1 (Figure S1I), indicating that defects in the replication checkpoint were not responsible for the loss of heterochromatin maintenance.
We further performed Taq polymerase-based random mutagenesis of the mrc1 + gene and isolated additional mrc1 mutant cells defective in heterochromatin maintenance but competent in transmitting replication checkpoint signals (Figure 1E).We isolated additional mrc1 missense and nonsense mutations, which localized downstream of the TQ motifs (Figure 1F).Cells carrying these mrc1 alleles formed white colonies on low adenine medium containing AHT and were resistant to HU, indicating that the C-terminal region of Mrc1 functions in heterochromatin maintenance independently of its replication checkpoint function.
To test whether defective heterochromatin maintenance in mrc1 mutant cells was due to changes in protein-protein interactions, we performed immunoprecipitation coupled with mass spectrometry (IP-MS) experiments of tandem affinity purification (TAP)-tagged Mrc1 proteins.The nonsense mutation at Mrc1-W620 produces a truncated protein that lacks the C-terminal phosphodegron, which stabilizes the mutant protein (Figure S1J). 80To generate cells that express similar levels of maintenance-competent and maintenance-defective Mrc1 proteins, we modified the endogenous mrc1 + gene to express TAPtagged phosphodegron-deficient Mrc1 (Mrc1-SSAA-TAP, maintenance-competent) and Mrc1-(1-620) (maintenance-defective) for IP-MS analysis (Figures S1J and S1K).As expected, IP-MS experiments showed that Mrc1 was associated with most replisome components (Figure 1G; Table S2). 51However, the association of replisome components with truncated Mrc1 was greatly reduced (Figure 1G).We obtained similar results by performing IP-MS from 33FLAG-tagged mrc1-SSAA, mrc1-W620STOP (maintenance-defective, checkpoint-defective), or mrc1-K769 STOP (maintenance-defective, checkpoint-competent) cells (Figure S1L; Table S3).However, in the Mrc1 IP-MS experiment, the spectral counts of the FACT subunit Spt16 and Pob3 were only mildly reduced (Figure 1G; Figure S1L), suggesting that Mrc1 associated with FACT independently of the replisome.In addition, IP-MS analysis of TAP-tagged Sld5, a component of the CMG helicase, from cells expressing Mrc1 or truncated Mrc1-(1-620), supported the observation that association of the truncated Mrc1 protein with the replisome was reduced (Figure 1H).These results raise the possibility that Mrc1 may help transfer parental histones by recruiting a FACT-histone complex or directly interacting with histones.
Structural predictions reveal a potential histone H3-H4 tetramer binding interface in Mrc1/CLASPIN Since several replisome components have been shown to bind histones through their unstructured charged regions together with the FACT complex, 81 we asked whether Mrc1 has a histone-binding region.Using template-free mode of AlphaFold-Multimer, [82][83][84][85][86][87] we identified a potential interaction interface between the S. pombe Mrc1-like domain (amino acids 701-837, Pfam database 88 ) and histone H3.1-H4 tetramer with a high confidence score (Figures 2A and 2B; Figures S2A and S2B).In the predicted structure, three alpha helices in the Mrc1-like domain (a1-3) form a brace that wraps around a histone H3.1-H4 tetramer in an asymmetric manner (Figure 2B).The fourth to sixth a helices (a4-6) occupied different relative positions in the five predicted models (Figure S2B), suggesting lower confidence in their interaction with the H3.1-H4 tetramer.The a1 and a3 of Mrc1-like domain were predicted to bind each of the two H3-H4 dimers, and the intervening a2 helix was predicted to simultaneously interact with both H4 subunits (Figure 2B).This distinctive interaction interface allows a single Mrc1-like domain to bind an entire H3-H4 tetramer, potentially serving to stabilize the H3-H4 tetramer during DNA replication.
To further explore the structural predictions, we performed additional AlphaFold-Multimer predictions and found that (1) full-length Mrc1 was predicted to interact with H3.1-H4 tetramer as well as centromere variant CENP-A/H4 (Cnp1/H4) tetramer specifically through the predicted Mrc1-like domain (Figures 2C and S2C-S2F), (2) the predicted Mrc1-histone-binding domain was conserved in eukaryotes, and homologs of the Mrc1-histone-binding domain from nine major model organisms representing fungi, animals, and plants were predicted to interact with H3.1-H4 tetramers with high confidence scores (Figure S2G), and (3) the interface predicted template modeling (ipTM) score between Mrc1/CLASPIN and H3.1-H4 tetramer,  or CENP-A/H4 tetramer from S. pombe, Drosophila melanogaster, and Homo sapiens, was the highest among all replisome components (Figure 2C), including the known histone H3-H4 binding proteins Spt16 and Mcm2, for which experimental structural information is available, and Pol1, Dpb3/ Dpb4, and Mcm10, for which no experimental structures are available but AlphaFold predicts relatively high confidence structures (Figures S3A-S3E).Together, these predictions suggest that Mrc1 contains a conserved histone H3-H4 tetramer binding domain.
We next aligned the predicted Mrc1-like domain-(H3.1-H4) 2 structure to the crystal structure of the nucleosome core particle (PDB: 1AOI) (Figures 2D and S3F). 26The alignment illustrated that the wrapping of the a1 helix of Mrc1-like domain around (H3-H4) 2 overlaps with nucleosomal DNA (approximately from the dyad to superhelical location-3, SHL-3, Figure S3G), and the binding of the Mrc1-like domain a2 and a3 helices to (H3-H4) 2 resembles the interactions of H2B-a2 and H2A C-terminal tail with (H3-H4) 2 in the nucleosome (Figure 2D; Figure S3G).Compared with H2B-a2, which only interacts with one of the H3-H4 dimers in the nucleosome, a2 of Mrc1-like domain is slightly tilted ($10.855 ), permitting it to interact with both H4s in a (H3-H4) 2 tetramer (Figure S3G).In addition to bearing a structural resemblance, the electrostatic surface of Mrc1-like domain resembles that of nucleosomal DNA, H2B-a2, and H2A's C-terminal tail (Figure 2D).The Mrc1-like domain therefore may associate with the H3-H4 tetramer in a manner that mimics nucleosome features and leads to partial displacement of nucleosomal DNA and at least one of the two H2A-H2B dimers.

Experimental validation of the predicted Mrc1 histonebinding domain
To experimentally test the AlphaFold-Multimer predicted interactions, we performed in vitro pull-down assays using recombinant glutathione S-transferase (GST)-tagged fragments of Mrc1 to examine their interactions with the histone H3-H4 tetramer.We found that Mrc1 fragment containing the Mrc1like domain (amino acids 601-900), but not other Mrc1 fragments, specifically pulled down histone H3-H4 under stringent binding and wash conditions (500 mM NaCl) (Figure 3A).Consistent with AlphaFold-Multimer predictions, Mrc1-like domain is only weakly associated with H2A-H2B (Figures S4A  and S4B).In addition, the Mrc1-like domain of S. cerevisiae Mrc1 and human CLASPIN both bound H3-H4; although relative to the human and S. pombe Mrc1-like domains, interaction of the S. cerevisiae Mrc1-like domain with H3-H4 was more salt-sensitive (Figures S4C and S4D).
To further test the AlphaFold predictions, we designed point mutations in the Mrc1-HBD, which are predicted to reduce its histone-binding activity.The predicted structure suggests that conserved amino acids with acidic side chains (Mrc1-E763,D767) in the middle of Mrc1-HBD a2 helix contact basic residues (H4-K91) in two histone H4s, whereas the two pockets formed by hydrophobic amino acids at both ends of Mrc1-HBD a2 helix accommodate hydrophobic residues in each of the two histone H4s (Figure 4A).GST pull-down assays under stringent binding conditions showed that mutating several amino acids (M755, F758, L774) in the hydrophobic pockets led to greatly reduced binding of Mrc1-HBD to H3-H4 (Figure 4B).Similarly, substitution of acidic residues in the middle of the Mrc1-HBD a2 helix with basic residues, Mrc1-E763R,D767K, greatly reduced binding to H3-H4 (Figure 4B).Mrc1-HBD a2 acidic and hydrophobic   amino acids are therefore required for complex formation with H3-H4.
0][91] At the pericentromeric DNA repeats, heterochromatin is continuously established by the RNAi pathway. 92,93Deletion of mrc1 + by itself had only a minor effect on H3K9me2 levels, suggesting that Mrc1 was not required for RNAi-dependent establishment of H3K9me2 (Figure 4F).In the absence of RNAi, residual H3K9me at pericentromeric repeats is epigenetically maintained by a Clr4 read/write-dependent mechanism. 7Combining a deletion of ago1 + (ago1D) with deletion of mrc1 + , or mrc1-HBD, or mrc1-3A (ago1D, mrc1D; ago1D, mrc1DHBD; ago1D, mrc1-3A) abolished the residual H3K9me2 (Figure 4F), indicating that Mrc1-HBD was required for epigenetic inheritance of pericentromeric H3K9me2.Together, these observations provide independent support for the structural predictions and further demonstrate that the Mrc1 histone-binding domain plays an important role in the maintenance of native heterochromatin in S. pombe.
We next tested the possible role of the histone-binding domain of Mrc1 (Mrc1-HBD) in gene silencing in S. cerevisiae, which diverged from S. pombe approximately 420-330 million years ago.We examined the effect of mrc1 deletion and mutations on silencing in a sensitized dual reporter S. cerevisiae strain, in which the TRP1 gene is located at the silent mating type HMR locus where the E silencer is deleted, and the URA3 gene is located near the left telomere of chromosome VII (Figures 5A  and 5B). 94Silencing of the TRP1 reporter inhibits growth on medium lacking tryptophan (ÀTrp), whereas silencing of the URA3 reporter allows cells to grow on medium containing 5-fluoroorotic acid (+FOA), which is toxic to URA3-expressing cells.In the absence of the E silencer, establishment of silencing by the I silencer is less efficient, and silencing may become more sensitive to the loss of parental histone transfer.Establishment of silencing at TEL-VIIL::URA3 locus is also less robust than silencing at other telomeres due to the engineered deletion of subtelomeric X 0 and Y 0 elements. 95Although this reporter system does not separate establishment and maintenance phases of silencing, it provides a sensitive assay for testing the possible effects of specific mutations on a chromatin-dependent silencing mechanism.
As shown in Figure 5C, mrc1D cells and cells with mutations in the conserved Mrc1-like domain (mrc1-D711-850, mrc1-D711-798) or in the Mrc1-HBD (mrc1-Da2, amino acids 760-790) were defective for silencing of the telomeric reporter gene URA3 to nearly the same extent as sir2D cells in which heterochromatin is not established.The HMR-ED::TRP1 locus was fully (G) eSPAN bias of parental histone surrogate H3K4me3 distribution around 162 origin of replication in wild-type (WT), mrc1-3A, mcm2-2A S. pombe cells.The shading of the bias line plot is the 95% confidence interval of mean value of at least two biological replicates, which is mean ± 2 folds of the standard error.See also Fang et al. 97 for WT and mcm2-2A eSPAN analysis.derepressed in Mrc1-HBD mutant cells but not in mrc1D cells.It has previously been shown that mrc1D cells have slightly shortened telomeres, 98 which is known to result in defective telomeric silencing but stronger silencing at the mating type locus. 99herefore, the robust silencing observed at the HMR-ED reporter in mrc1D cells may result from redistribution of limiting silencing proteins to the HMR-ED locus, allowing more efficient I silencerdependent establishment, masking the mrc1D maintenance defect.Deletion of DPB3 (dpb3D), which has an established role in parental histone transfer to the leading strand, 16 also had no effect on silencing of the HMR-ED::TRP1 locus, but silencing at this locus was lost in mrc1D dpb3D double mutant cells (Figure 5C).This suggests that at the S. cerevisiae HMR-ED::TRP1 locus Mrc1 and Dpb3 may play redundant roles in the leading strand histone transfer pathway.We conclude that the histone-binding domain of Mrc1 plays an evolutionarily conserved role in maintaining silent chromatin domains.

Mrc1 is required for parental histone maintenance following DNA replication
To test whether the histone-binding activity in Mrc1 contributes to the symmetric inheritance of parental histones, we conducted enrichment and sequencing of protein-associated nascent DNA (eSPAN) using histone modifications H3K4me3 and H3K56ac as surrogates for parental and new histones, respectively, in S. cerevisiae and S. pombe cells (Figure S5A).As expected, 16 in wild-type S. cerevisiae cells, we observed no apparent bias of parental and new histone inheritance at daughter strands around 139 early replication origin regions, indicating symmetrical distribution of parental histones at both strands (Figures 5D, S5B, S5C, and S5H).By contrast, mrc1D and mrc1-like domainD (mrc1-D711-850) cells displayed weak preferential transfer of parental histones (H3K4me3) toward the lagging strand (Figures 5D, S5B, S5C, and S5H).As controls, dpb3D cells had a strong eSPAN H3K4me3 bias toward the lagging strand, which was enhanced in dpb3D, mrc1D, and dpb3, mrc1-like domainD double mutant cells (Figure 5D).New histones (H3K56ac), on the other hand, showed a slight bias toward the leading strands in the mutant cells, suggesting that defects in the transfer of parental histones to the leading strand were partially compensated by new histone deposition (Figures 5D, S5D, and S5I).Consistent with an important role for Mrc1 in governing symmetrical parental histone transfer, the strong leading strand bias of mcm2-3A cells 14,15 was completely reversed in mcm2-3A, mrc1D double mutant cells (Figure 5E).Loss of the entire Mrc1 protein may therefore lead to inefficient recycling of parental histones and suppresses the biased H3K4me3 eSPAN ratios.
Because Mrc1 makes extensive contacts with other replisome components [100][101][102] and Mrc1-like domain contains regions that do not directly interact with histones, deletion of the entire Mrc1 or Mrc1-like domain may impact parental histone transfer ratios independently of the histone-binding activity of Mrc1.To specifically test whether Mrc1-HBD has intrinsic histone transfer bias, we performed eSPAN experiments using mrc1 mutations that abolish histone binding without affecting interactions with the replisome: mrc1-Da2 in S. cerevisiae and mrc1-3A in S. pombe.In support of a specific effect on histone binding, MS analysis of Sld5-TAP IPs from mrc1 + and mrc1-3A S. pombe cells showed that Mrc1-3A remained associated with the replisome and did not affect the association of other replisome proteins with Sld5 (Figure S6A).Surprisingly, unlike mrc1D or mrc1like domainD, mrc1-Da2 S. cerevisiae cells had no apparent strand bias patterns for H3K4me3 or H3K56ac (Figure 5F; Figures S5E-S5I).Consistent with the S. cerevisiae results, eSPAN analysis in mrc1-3A S. pombe cells showed no apparent strand bias for H3K4me3, whereas control mcm2-2A cells showed a strong expected leading strand eSPAN bias (Figure 5G; Figure S5J).Therefore, eSPAN analysis of Mrc1 histone-binding mutant cells in both S. cerevisiae and S. pombe suggests that mutations in Mrc1-HBD do not affect symmetrical histone transfer.
Based on the above results, we hypothesize that loss of heterochromatin maintenance in Mrc1 histone-binding mutant cells results from reduced parental histone transfer to both daughter DNA strands.Consistent with this hypothesis, eSPAN experiments in S. pombe indicated the H3K4me3 density around the origins of replication at both the leading and lagging strands is significantly reduced in mrc1-3A cells (lagging strand reduced 35.0%and leading strand reduced 31.8%,p value < 0.001) (Figure 5H).We further used ChIP to examine the maintenance of H3K9me2 at the 10XtetO-ade6 + locus in cells that carried a TetR-Clr4-DCD to establish H3K9me2 at 10XtetO locus, lacked endogenous Clr4 methyltransferase, and carried a cdc25-22 temperature-sensitive allele allowing cell cycle arrest at late G2 phase at 36 C and release of synchronized cells from the arrest at 25 C (tetR-clr4-DCD, clr4D, cdc25-22, Figure 5H).tetR-clr4-DCD, clr4D cells are read-write deficient, allowing us to establish H3K9me2 in cell cycle-synchronized cells and then track its recycling following the release of TetR-Clr4-DCD and progression through S phase (Figure 5I).Both mrc1-3A and mcm2-3A cells maintained less H3K9me2 6 h after the release of TetR-Clr4-DCD and progression through the cell cycle, indicating that they were defective in recycling parental histones (Figure 5J).These results suggest that Mrc1-HBD distributes histones to both the leading and lagging strand transfer pathways without affecting symmetrical parental histone transfer.
Distribution of FACT binding sites on the replisome Since Mrc1 associates with the FACT complex independently of the replisome (Figure 1G), 103 and previous works showed that the N terminus of Mcm2 binds to histones together with FACT, 32 we hypothesize that Mrc1 and other histone-binding proteins in the replisome can co-chaperone histones with FACT.To gain additional insight into the interactions of FACT with the replisome, we performed pairwise AlphaFold-Multimer predictions between FACT subunits and replisome components (Figure S6B).Consistent with the IP-MS results, AlphaFold-Multimer predicted two FACT binding domains (FBDs) in Mrc1, which we confirmed by GST-pull-down assays (Figures S6C  and S6D).Mrc1-FBD1 (amino acids 134-168) interacts with the Spt16 middle domain (MD) (amino acids 664-930) (Figures S6E-S6G).Mrc1-FBD2 (amino acids 513-540) is located near the Mrc1-HBD (amino acids 708-809) and interacts with the Spt16 N-terminal domain (NTD) (amino acids 2-437) (Figures S6H-S6J).AlphaFold-Multimer structural predictions show that the Mrc1-HBD may engage an H3-H4 tetramer bound to the Spt16 MD, supporting the idea that Mrc1, like Mcm2, may co-chaperone histones together with FACT (Figures S6F and  S6I).However, deletions of Mrc1-FBDs had no effect on heterochromatin maintenance, suggesting that other FACT binding sites on the replisome may compensate for the loss of contacts with Mrc1 in vivo.
AlphaFold-Multimer also identified potential interaction interfaces between Spt16 and the Swi1 subunit of the FPC and the Pol1 subunit of DNA polymerase a (Figures 6A-6E; Figure S6B).The predicted Swi1-Spt16 interaction is mediated by the C-terminal domain of Swi1 (Swi1-CTD) and the Spt16-NTD (Figures 6A and 6B; Figure S7A), which is conserved in S. cerevisiae and is one of the previously reported Swi1 domains shown to interact with FACT in pull-down experiments. 60Deletion of Swi1-CTD (swi1-D832-894) abolished heterochromatin maintenance, suggesting that FACT recruitment via Swi1 may play a role in parental histone transfer (Figure 6C).

DISCUSSION
In this study, we identify the Mrc1/CLASPIN subunit of the FPC as an H3-H4 tetramer chaperone critical for parental histone maintenance during DNA replication and heterochromatin inheritance.Our findings suggest that Mrc1/CLASPIN together with FACT and other replisome components form a network of chaperones that coordinate the transfer of intact parental histone H3-H4 tetramers to newly replicated DNA.The location of the Mrc1 histone-binding domain and the FPC on the replisome and the requirement for Mrc1 in parental histone transfer to both daughter DNA strands suggest that Mrc1-HBD acts as part of a distribution center for the initial capture and transfer of histones to the leading and lagging strand pathways (Figure 7; Figures S7J-S7P).
Our findings suggest broad roles for Mrc1 and Mrc1-HBD in parental histone transfer to newly replicated DNA.eSPAN analysis of cells carrying a full deletion of mrc1 + (mrc1D) or deletions of the Mrc1-like domains extending beyond its histone-binding domain display a weak bias for parental histone transfer to the lagging strand in S. cerevisiae, suggesting that symmetrical histone transfer requires Mrc1.Larger Mrc1 deletions furthermore greatly enhance the lagging strand bias of dpb3D cells, suggesting that Mrc1 and Dpb3 function together in the leading strand transfer pathway.However, S. cerevisiae cells with a deletion of the Mrc1-a2, which specifically disrupts H3-H4 binding, do not affect the eSPAN bias ratios.Similarly, S. pombe Mrc1 mutations that specifically disrupt H3-H4 binding (mrc1-3A) do not affect eSPAN bias ratios but greatly reduce the maintenance of parental H3K9me after DNA replication.These observations suggest distinct roles for the Mrc1-HBD and other Mrc1 domains in parental histone transfer that include roles for Mrc1 in coordinating the activities of other replisome components to ensure symmetrical parental histone transfer (via domains outside its HBD) 105 and direct distribution of parental histone to both the leading and lagging strand pathways (via its HBD).
The available cryo-EM structures of the replisome 96,106 and AlphaFold structural predictions suggest that Mrc1 makes extensive interactions with other replisome components and allows us to pinpoint the location of Mrc1-HBD (Figure 7A; Figures S7J-S7P).The interactions of Mrc1 regions adjacent to its HBD with the Cdc45/Mcm2 components of the replicative helicase suggest that the Mrc1-HBD is located at a central position on the replisome from which it may act as a distribution site for the transfer of parental H3-H4 tetramers to either the leading or the lagging strands (Figure 7; Figures S7J-S7P).Beyond its HBD, Mrc1 interacts with multiple components of the replisome, including other subunits of the FPC, Cdc45, Mcm2, and the interacting region was identified by AlphaFold-Multimer and is consistent with previous biochemical results. 102The newly identified histone-binding region is highlighted in pink and the Cdc45/Mcm2(NTD) interacting region is highlighted in red.Bottom, the predicted structure of Mrc1-like domain/(H3-H4) 2 /Cdc45/ Mcm2(NTD) was aligned to the cryo-EM structure (PDB: 8B9C) via the Mrc1-like domain a5 helix.See Figures S7K-S7P for alignment details. (B) Model for DNA replication-coupled directional parental histone transfer with FACT acting as a mobile chaperone.P, parental site; D, distribution site; LD1, leading strand site 1; LG1 and LG2, lagging strand sites.See text for details.See also Figure S8.catalytic subunit of DNA polymerase ε 96,100-102 (Figure 7A; Table S6).The complete absence of Mrc1 may therefore alter the structure of the replisome in a way that globally disrupts strand-specific parental histone transfer.In this model, Mrc1 would act as a key modulator of the overall replisome conformation ensuring that multiple histone-binding domains are properly orientated to achieve symmetrical parental histone transfer.This model also provides an explanation for distinct phenotypes of mutations in the Mrc1-HBD compared with the deletion of the entire Mrc1 or mutations outside its HBD.It also raises the exciting possibility that regulation of Mrc1 interactions may contribute to biased parental histone transfer at specialized replication forks or cells. 107,108ompared with other histone-binding replisome components, Mrc1 contains a binding interface with the entire H3-H4 tetramer through physical properties that resemble nucleosomal components that bind to the H3-H4 tetramer in the nucleosome core particle.This mode of H3-H4 binding may be critical for the transfer of intact H3-H4 tetramers to newly synthesized DNA.By contrast, experimental 33,34 and predicted structures suggest that Mcm2, Pol1, and Dpb3/Dpb4 only bind to H3-H4 dimers (Figures S3A-S3E) and are therefore likely to have a more stringent requirement for the FACT complex in transporting H3-H4 tetramers.Several recent studies show that FACT favors binding to destabilized over intact nucleosome substrates 47,48 and is required for chromatin replication in vitro. 52Parental nucleosome disassembly in response to the force exerted by the replicative CMG helicase may be facilitated by binding of FACT to the partially disassembled nucleosome (Figure 7B), similar to the association of FACT with partially disrupted nucleosomes during transcription elongation. 49,50In addition, FACT has domains that interact with the catalytic subunit of DNA polymerase a Pol1, 57 RPA, 37,59 Mcm2-7 complex, 32,109 Tof1/Swi1, 60 and Mrc1 (this study).The requirement for the FACT binding sites on Swi1 and Pol1 in epigenetic maintenance of heterochromatin supports the idea that FACT-replisome interactions contribute to parental histone recycling.
Our analysis of the locations of histone-binding domains on the structure of the replisome 96,100,101,106 allows us to propose stepwise pathways for the transfer of parental histones to newly replicated DNA (Figure 7B).We propose that the parental nucleosome is destabilized by the CMG helicase, leading to recruitment of the FACT complex and further nucleosome disassembly 46,48 (Figure 7B, parental or P site).FACT captures parental histones from the P site and is then recruited to the replisome through its interaction with the Swi1 subunit of the FPC (Figure 7B).Since Swi1 interacts with Mrc1, Mcm2, FACT, 60 and histones, and Mrc1 contributes to parental histone transfer to both daughter DNA strands, we propose that Swi1 and Mrc1-HBD forms a distribution hub (D site) for transfer of the FACT-H3-H4 complex to the leading or lagging strands (Figure 7B).Leaving the D site, the FACT-histone complex may be captured by Dpb3-Dpb4 (leading site 1 [LD1 site]) for deposition onto the newly synthesized leading DNA strand.For the lagging strand pathway, the FACT-H3-H4 complex would be transferred from the D site to the Mcm2 histone-binding domain (lagging site 1 [LG1 site]) and to Pol1 (LG2 site) for deposition of histones onto the lagging strand (Figure 7B).The transfer mechanism is dynamic and may rely on intermediate states in which Mrc1-HBD directly hands off (H3-H4) 2 to other his-tone-binding proteins in the replisome along the leading or lagging strand pathways.This idea is supported by the apparent extended and the likely flexible structure of Mrc1 and AlphaFold predictions, suggesting that Mrc1 and each of the histone-binding proteins along the leading and lagging strands can simultaneously associate with (H3-H4) 2 (Figure S8).

Limitations of the study
The complexity of the replisome, together with the large distances that parental histone must travel from the front of the replisome to newly replicated DNA, suggests that our understanding of the transfer pathway is still rudimentary.Future experiments are required to understand how the Swi1-Mrc1 hub coordinates the symmetrical and directional transfer of parental histones to the leading and lagging strand binding sites before their deposition on newly synthesized DNA.The proposed order of the binding and transfer events, as well as the AlphaFold predicted structures of intermediate parental histone transfer states, also require further experimental demonstration.

Lead contact
Further information and requests for reagents or resources should be directed to and will be fulfilled by the lead contact, Danesh Moazed (danesh@hms.harvard.edu).The materials generated in this study will be provided without restriction.

Materials availability
Resources and materials generated in this study are available upon request and the request should be directed to lead contact Danesh Moazed.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Plasmids
All plasmids used in this study were generated using Gibson Assembly, 110 except for CRISPR-based genome editing plasmids used for construction of some of the S. pombe mutant cells, which were generated using Golden Gate ligation. 111Antibiotics resistant gene-containing plasmids pFA6a-kanMX6, natMX6, hphMX6, bsdMX were used as the backbones to generate plasmids to amplify PCR fragments for yeast transformation.pGEX-6p-1 containing GST followed by the 3C protease cleavage site was used as the backbone to generate GST-fusion protein constructs for recombinant protein expression and purification.
Yeast strains All S. pombe and S. cerevisiae strains were generated using homologous recombination-based mutagenesis with PCR amplified fragments that carried homology arms and desired mutations 112,113 except for swi3D, rfa3D and ctf18D S. pombe strains, which were generated using CRISPR-Cas9. 111All S. pombe and S. cerevisiae strains used in this study are listed in Table S1, respectively.gRNAs used to delete swi3 + , rfa3 + , ctf18 + are listed in Table S1.

Yeast reporter assays
For heterochromatin maintenance and replication stress assays, S. pombe cells were cultured in YES media overnight and then diluted to 1.0x10 5 cells/mL (OD 600 =1.0, Nanodrop).Cells were washed with sterile water and resuspended to 4x10 5 cells/mL (OD 600 =4.0, Nanodrop).Serial dilutions (1, 1:10, 1:100, 1:1000, 1:10000) of cells were then spotted on YE (low adenine), YE+10 mM anhydrotetracycline (AHT, Cayman chemical), or YES+5 mM hydroxyurea (Sigma-Aldrich) plates to assay heterochromatin establishment, maintenance, and replication stress, respectively.The plates were photographed after incubation at 32 C for 3 days.For DNA-sequence dependent heterochromatin maintenance assays, S. pombe cells were prepared as above and plated on YES, EMMc-Ura (EMM powder, Sunrise Science Products), or EMMc+FOA (5-FOA, Goldbio) plates to assay heterochromatin establishment and maintenance at the mating type locus.To quantify the percentage of silent colonies in the heterochromatin maintenance assay, 60 mL 1:1000 dilute cells from the density of OD 600 =1 were plated on YE+AHT plates.For heterochromatin spreading assay, S. pombe cells were prepared as above and plated on YE plate.For S. cerevisiae gene silencing assay, cells were cultured in YEPD+Ade+Trp medium overnight and diluted to OD 600 =1.0 (Nanodrop).Cells were washed with water and resuspend to 4x10 5 cells/mL (OD 600 =4.0, Nanodrop).Serial dilutions (1, 1:10, 1:100, 1:1000, 1:10000) of cells were then spotted on YEPD+Ade+Trp, SC-Trp, SC+FOA, or YEPD+Ade+Trp+50 mM HU plates to assay cell growth, reporter gene silencing at the mating type locus and telomere, and replication stress phenotype, respectively.The plates were photographed after incubation at 30 C for 2 days.Images were captured by Nikon D70 under the control of Nikon Camera Control Pro.Global adjustment of contrast and saturation of the images were conducted by Adobe Lightroom for the presentation.

Chromatin immunoprecipitation
To prepare ChIP samples, S. pombe cells were cultured in YES medium overnight and diluted to OD 600 = 0.2 in YES medium and processed for ChIP as previously described 114 with modifications.For heterochromatin maintenance phase experiment, S. pombe cells were cultured with 10 mM AHT for 24 hours.For cell cycle synchronization experiment, cdc25-22 S. pombe cells were first cultured at 25 C in mid log-phase, then transferred to 36 C culture for 4 hours to arrest at late G2 phase, and then immediately cool down in water bath at 25 C supplemented with 10 mM AHT and cultured for another 6 hours at 25 C to release from late G2 phase and resume cell cycle.After reaching OD 600 =2$3, cells were crosslinked in 1% methanol-free formaldehyde (16% w/v formaldehyde, ThermoFisher) for 15 min at room temperature, followed by quenching using 100 mM glycine for 5 min at room temperature.Cells were then pelleted by centrifuging at 5,000 rpm for 1 min at 4 C, washed with 1 mL cold TBS (20 mM Tris, 150 mM NaCl) buffer, flash frozen in liquid nitrogen, and stored at -80 C. Frozen cell pellets were resuspended in ChIP lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 1 mM EDTA, 1 mM PMSF supplemented with cOmplete protease inhibitor cocktail(Sigma-Aldrich)). 1 mL acid-wash glass beads were added and cells were lysed with MagNA Lyser (Roche) using the program: 3 rounds of 90 s with 4,500 rpm and 1 round of 45 s with 5,000 rpm.Cells were placed in ice-water slush for 1 min to cool down in between each cycle.The lysate was then resuspended to 1 mL and sonicated in millTUBE 1 mL AFA fiber (Covaris) on Covaris E220 evolution sonicator at 4 C using the program: 5% duty cycle, 140 PIP, 200 cycle per burst for 12 min.The lysate was then centrifuged at 13,200 rpm for 15 min at 4 C.The supernatant was collected, 5% of which is saved as input.The remainder of each sample was incubated with Dynabeads protein A (Invitrogen) conjugated anti-H3K9me2 antibody (Abcam) at 4 C for 3 hours.30 mL protein A magnetic beads were incubated with 2 mg anti-H3K9me2 antibody at 4 C for 1 hour and then added to each sample.After incubation, magnetic beads were collected using a magnetic stand and washed with ChIP lysis buffer three times and with pre-chilled TE once.Magnetic beads were then eluted with 100 mL ChIP elution buffer A (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) and 150 mL ChIP elution buffer B (TE with 0.67% SDS) for 5 min at 65 C with 1,400 rpm on an Eppendorf Thermomixer F1.5.Eluted fractions were combined and incubated at 65 C overnight to reverse crosslinks.Samples were then treated with ChIP protein digestion buffer containing 3 mg proteinase K (Roche), 100 mM LiCl, and 5 mg glycogen (Roche) in TE at 55 C for 1 hour.ChIP and input DNA were then purified using phenol-chloroform extraction followed by ethanol precipitation.Percent of input of ChIP DNA was then analyzed by quantitative PCR of input and ChIP DNA on Applied Biosystems QuantStudio 7 flex.All qPCR primers used for ChIP experiments are listed in Table S1.

Immunoprecipitation
Immunoprecipitations of replisome factors were carried as described 32 with modifications.S. pombe cells were cultured overnight at 32 C in YES medium, diluted to OD 600 =0.05 in YES medium, and incubated in a shaker at 32 C for 14 hours.1x10 10 cells were harvested by centrifuging at 5,000 rpm for 10 min at 4 C and cell pellets were washed once with 25 mL prechilled TBS buffer.The cell pellets were weighed and resuspended in 1/5 volume of resuspension buffer (20 mM HEPES-KOH pH 7.5, 100 mM KOAc, 5 mM Mg(OAc) 2 , 0.25% Triton X-100, 1 mM EDTA, 10% (v/v) glycerol).Cell resuspensions were then added into liquid nitrogen dropwise to form frozen yeast popcorn.Cells were then broken by grinding the yeast popcorn using Freezer/Mill 6875D with 12 cycles of 90 s vortex, 2 min cool (speed: 10 CPS) and stored in -80 C. Ground yeast powder was resuspended in lysis buffer (20 mM HEPES-KOH, pH 7.5, 100 mM KOAc, 5 mM Mg(OAc) 2 , 0.25% Triton X-100, 5 mM NaF, 5 mM b-glycerophosphate, 1 mM EDTA, 1 mM PMSF, 1 mM DTT, 10% (v/v) glycerol, supplemented with Roche cOmplete protease inhibitor and protease inhibitor cocktail (Sigma, P8215)), treated with 1000 U/mL Benzonase (Santa Cruz Biotechnology, Catalog No. sc-391121C) for 1 hour at 4 C.The lysate was centrifuged for 3 min and then 15 min at 13,200 rpm.The supernatant was then incubated with antibodies crosslinked with magnetic beads at 4 C for 3 hours.Magnetic beads were collected on a magnetic stand, washed with lysis buffer four times, and eluted using 0.5 M NH 4 OH at 37 C for 20 min.Elutions from beads were then dried in a speed vacuum and analyzed by SDS-PAGE, western blot and mass spectrometry.For TAP immunoprecipitation of Mrc1 or Sld5 proteins, Rabbit IgG (Sigma, 15006) was conjugated to Dynabeads M270 Epoxy (Invitrogen, 14302D) and stored in 1xPBS+0.02%sodium azide at 4 C before being used for immunoprecipitation.For FLAG immunoprecipitation of Mrc1 proteins, anti-FLAG M2 antibody (Sigma, F1804) was incubated with Dynabeads Protein G (Invitrogen, 10004D) overnight before being used for immunoprecipitation.All antibody-conjugated magnetic beads used in immunoprecipitation were first crosslinked with 14.8 mM dimethyl pimelimidate (DMP, Invitrogen, 21667) in 10 bead-volume of crosslinking buffer (0.2 M sodium borate, pH 9) at room temperature for 30 min, followed by quenching using 10 bead-volume of 0.2 M ethanolamine (Sigma, E9508) at room temperature for 90 min.
The spectral counts of proteins identified by mass spectrometry are listed in Tables S2, S3, S4, and S5.
Label-free mass spectrometry Label-free mass spectrometry analysis was performed using on-bead digestion.In solution digestion was performed on beads from immunoprecipitations. 20 ml of 8 M urea (Sigma-Aldrich), 100 mM EPPS (Sigma-Aldrich) pH 8.5 were added to the beads.5 mM TCEP was added, and the mixture was incubated for 15 min at room temperature.10 mM of iodoacetamide was then added for 15min at room temperature in the dark.15 mM DTT was then added to consume any unreacted iodoacetamide.180ml of 100 mM EPPS pH 8.5 was added to reduce the urea concentration to <1 M, followed by the addition of 1 mg of trypsin (Promega) and incubated at 37 C for 6 h.The solution was acidified with 2% formic acid and the digested peptides were desalted via StageTip, dried via vacuum centrifugation, and reconstituted in 5% acetonitrile, 5% formic acid for LC-MS/MS processing.
Taq-based gene-targeted random mutagenesis Yeast strain SPY9210 (mrc1-W620STOP-ura4/hphMX6) was used for the mutagenesis.In brief, cells were transformed with fulllength Mrc1 fragments generated by Taq polymerase-mediated PCR to replace the missing C terminus of mrc1, ura4-hphMX6 drug cassette to generate a complete mrc1 allele with random mutations generated by Taq polymerase during PCR.Transformants were selected on FOA plates with two rounds of replica plates.Transformants were then plated on YE, YE+10 mM AHT, YES+5 mM HU and screened for colonies that display red color on YE plates, white color on YE+AHT plates, and viability on YES+HU plates.Candidate colonies were streaked on the YE+AHT plates for single colony purification and candidates with variegated color displayed on the YE+AHT plates were discarded.Cells grown from a single colony from individual candidates were then assayed again on YE, YE+AHT, YE+HU plates with serial dilutions to confirm maintenance-specific defects.The entire mrc1 gene from each candidate was amplified, followed by Sanger sequencing to identify mutations.

Purification of S. pombe FACT complex
The FACT complex was purified as described previously 48 with modifications.Endogenously FACT was purified from Pob3-TAP tagged S. pombe strain and overexpressed FACT was purified from S. pombe strain overexpressing Spt16, Pob3-TAP driven by nmt1 promoter in EMMc media.For endogenous FACT purification, Yeast popcorn from 1 L cell culture was prepared as described above for replisome purifications.The yeast popcorn was resuspended in lysis buffer-FE (20 mM HEPES-KOH pH 7.5, 600 mM KOAc, 5 mM Mg(OAc) 2 , 0.01% CHAPS (anatrace), 0.01% octyl-glucoside (anatrace), 1 mM EDTA, 1 mM PMSF, 1 mM DTT, 10% (v/v) glycerol supplemented with Roche cOmplete protease inhibitor).Supernatant was prepared as described above for replisome purifications and incubated with IgG-conjugated Dynabeads or IgG Sepharose 6 Fast Flow affinity resin (Cytiva) at 4 C for 2 hours with rotation.The magnetic beads or resin were collected and wash with lysis buffer four times.The beads were then equilibrated in elution buffer (20 mM HEPES-KOH pH 7.5, 150 mM KOAc. 5 mM Mg(OAc) 2 , 1 mM EDTA, 1 mM PMSF, 1 mM DTT, 10% (v/v) glycerol).FACT complex was eluted from magnetic beads or resin with TEV protease at room temperature for 1 hour with rotation.For overexpressed FACT purification, yeast popcorn was lysed in lysis buffer-FOE (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5 mM MgCl 2 , 0.01% CHAPS, 0.01% octyl-glcoside, 1 mM EDTA, 1 mM PMSF, 10% glycerol with Roche cOmplete protease inhibitor).After TEV cleavage, the eluted complex was subjected to anion exchange chromatography HiTrap Q HP 1 mL in the gradient of NaCl from 100 mM to 1 M. Peak fractions containing FACT complex was further purified in a size exclusion chromatography with Superdex 200 increase 10/300 GL.Purified complex was then analyzed by SDS-PAGE, silver staining, and western blotting.Anti-calmodulin binding protein epitope tag antibody (1:5000 dilution, Sigma) was used to detect Pob3 subunit by western blotting.
GST pull-down assay BL21-CodonPlus competent cells were transformed with pGEX-6p-1 vectors expressing the fusion of GST-tag and fragments of S. pombe Mrc1 protein, including Mrc1-like domain, Pol1-N-terminal extension (NTE) and its mutants, S. cerevisiae Mrc1-like domain, or human Mrc1-like domain in CLASPIN connected by 3C protease cleavage site using protocols from Agilent.BL21-CodonPlus competent cells carrying pGEX-6p-1 vectors were cultured in 50 mL LB media with 100 mg/mL ampicillin and 25 mg/mL chloramphenicol and induced with 2% ethanol and 0.2 mM IPTG (AmericanBio) at 20 C for 4 hours with shaking at 220 rpm starting with OD 600 =0.5$0.9.Cells are collected by centrifugation at 7,000 rpm for 10 min at 4 C. Cell pellets were resuspended in B-PER Complete Bacterial Protein Extraction Reagent (ThermoFisher) supplemented with 900 mM NaCl, 1 mM PMSF, 1 mM DTT and 1 mM EDTA and lysed at 4 C for 30 min with rotation.The lysate was then centrifuged at 15,000 rpm for 20 min at 4 C. Supernatant was collected, diluted with one volume of Wash/Equilibrium buffer (20 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 0.02% Triton X-100, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10% (v/v) glycerol) and incubated with 20 mL Pierce Glutathione Sepharose Magnetic Agarose Beads (ThermoFisher) at 4 C for 1 h with rotation.The magnetic beads were collected on a magnetic stand and washed with Wash/Equilibrium buffer four times.To test the interaction between Mrc1-like domain and H3-H4 tetramer, FACT complex, the magnetic agarose beads was then equilibrated in the Binding buffer and incubated with in vitro reconstituted H3-H4 tetramer (Binding buffer for H3-H4 tetramer: 20 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 0.02% Triton X-100, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mg/mL insulin, 10% (v/v) glycerol) or purified fission yeast FACT (Binding buffer for FACT complex: 20 mM HEPES-KOH pH 7.5, 150 mM KOAc, 5 mM Mg(OAc) 2 , 1 mM EDTA, 1 mM EDTA, 10% (v/v) glycerol), which was endogenously expressed, at 4 C for 1 h with rotation.For the GST-pull-down experiments to test the interaction between Pol1-NTE domain with FACT complex, H3-H4 tetramer and Mcl1-CTD domain, the wildtype and mutant GST-Pol1-NTE proteins were immobilized on the magnetic beads, equilibrated in the Pol1 binding buffer (PB buffer: 20 mM Tris-HCl, pH 7.5, 0.02% Triton X-100, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mg/mL insulin, 10% (v/v) glycerol) and incubated with overexpressed fission yeast FACT in PB buffer + 100 mM NaCl, H3-H4 tetramer in PB buffer + 300 mM NaCl, or Mcl1-CTD domain in PB buffer + 150 mM NaCl at 4 C for 1 h with mixing.The magnetic beads were collected on a magnetic stand and washed with Binding buffer for six times.The beads were then boiled in sample buffer and analyzed by SDS-PAGE and Coomassie blue stain, silver stain, and western blot.
Enrichment and sequencing of protein-associated nascent DNA The eSPAN assay in S. cerevisiae was adapted from previous methods with minor modifications. 14,16S. cerevisiae yeast cells were cultured in YPD medium at 30 C and 180 rpm shaking until they reached the mid-log phase (OD 600 =0.4-0.5).To arrest cells at the G1 phase, they were treated with 5 mg/mL a-factor (Chinese Peptide Company) twice at 25 C and 180 rpm for one hour each time.Subsequently, the cells were pelleted by centrifugation at 2,500 rpm for 5 min at 4 C, washed three times with cold ddH 2 O, and then released into fresh YPD medium with 0.4 mg/mL BrdU (Sigma-Aldrich) at 23 C for 40 minutes to label newly synthesized DNA.Afterwards, the cells were crosslinked with 1% (w/v) paraformaldehyde (Sigma-Aldrich) at 25 C and with gentle rotation at 180 rpm for 20 minutes, followed by quenching with 125 mM glycine (Amresco) at 25 C and with gentle rotation at 180 rpm for 5 minutes.The resulting cells were then pelleted, washed twice with cold TBS buffer (0.1 mM PMSF freshly added), and once with cold Buffer Z (1.2 M sorbitol, 50 mM Tris-HCl pH 7.4).The cells were resuspended in 8.7 mL Buffer Z (10 mM b-mercaptoethanol freshly added), and digested by adding 214 mL 5 mg/mL Zymolase (nacalai tesque) with incubation at 28 C and 100 rpm for approximately 35 minutes.The efficiency of digestion was checked by measuring the OD 600 in 1% SDS, which should decrease to less than 10% of that pre-digestion value.The spheroplasts were collected by centrifugation, and the supernatant was aspirated.The pellet was gently resuspended in 1.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl 2 , 1 mM CaCl 2 , with 0.5 mM Spermidine, 0.007% (v/v) b-mercaptoethanol and 0.075% (v/v) NP-40 (Thermo) added freshly), and the resuspended pellet was divided into 4 equal parts, with each part containing 400 mL.The appropriate amount of MNase (Worthington, LS004797) was added to each part, and the reaction mixtures were incubated at 37 C for 20 min to digest the chromatin into mainly mono-and di-nucleosome.The reaction was stopped by 8 mL 0.5 M EDTA (pH 8.0).Subsequently, 100 mL of 53 ChIP lysis buffer (250 mM HEPES-KOH pH 7.5, 700 mM NaCl, 5 mM EDTA pH 8.0, 5% (v/v) Triton X-100, 0.5% (w/v) Sodium deoxycholate, with 5 mM PMSF, 1.25 mg/mL pefabloc, 5 mg/mL bacitracin and 5 mM benzamidine added freshly) was added to the reaction mixtures, followed by 30 min of incubation on ice.The lysate was spun down twice at 10,800 rpm for 5 min and 15 min, respectively, at 4 C.The supernatant was collected and used for DNA extraction.
For each experiment, 50 mL of the supernatant was used as input, and 800 mL was used for ChIP against H3K4me3 or H3K56ac antibodies.For the ChIP assay, each sample was incubated with 0.6 ng anti-H3K4me3 antibody (Abcam) or 0.5 mL anti-H3K56ac antibody at 4 C for 12 hours, followed by incubation with 20 mL pre-washed protein G Sepharose agarose beads (GE Healthcare) at 4 C for 2 hours.The reaction mixtures were then washed extensively as below with 1 mL buffer per sample each time, and spun down at 2,500 rpm for 1 min at 4 C: 1xChIP lysis buffer (with 0.1 mM PMSF), once; 1xChIP lysis buffer, 5 min of incubation at 4 C, twice; 1xChIP lysis buffer (with 0.5 M NaCl), once; 1xChIP lysis buffer (with 0.5 M NaCl), 5 min of incubation at 4 C, once; Tris/LiCl buffer, once; Tris/LiCl buffer, 5 min of incubation at 4 C, once; Tris/EDTA buffer, twice.After washing, any remaining liquid was removed with fine syringe needles.Both the input and ChIP samples were reverse-crosslinked with chelex-100 (Bio-Rad).50 mL 20% (w/v) chelex-100 is added to each sample, followed by 10 min at 100 C.
After cool-down, 5 mL 20 mg/mL Proteinase K (Invitrogen) was added, with 30 min of incubation at 55 C, followed by 10 min at 100 C. The sample was then spun down at 14,000 rpm for 1 min and the supernatant was saved with 75 mL for the input sample and 25 mL for the ChIP sample.After adding 50 mL 2xTE to the original tube, the resulting DNA sample was cleared at 14,000 rpm for 1 min and mixed with the supernatant collected before.For the ChIP sample, 35 mL 1xTE was added to the original tube and cleared at 14,000 rpm for 1 min.The resulting 35 mL supernatant was saved and mixed with the supernatant collected before.For both the input and ChIP samples, 90 mL of the supernatant was used for BrdU-IP to obtain BrdU-IP and eSPAN samples, respectively.
For BrdU-IP, each sample was boiled at 100 C for 5 min and then snap-cooled in ice water for 5 min to get denatured singlestranded DNA.The reaction mixtures with anti-BrdU antibodies were prepared freshly with 800 mL cold BrdU-IP buffer (1xPBS, 0.0625% TritonX-100), 0.36 mL anti-BrdU antibody (BD Biosciences) and 0.3 mL 20 mg/mL E. coli tRNA (Roche) for each sample.The denatured sample was then mixed with 10 mL 10XPBS and 800 mL reaction mix, followed by 2 hours of incubation at 4 C.The reaction mixtures were then incubated with 15 mL pre-washed protein G beads for 2 hours at 4 C, followed with extensive wash as below: cold BrdU-IP buffer, 4-5 min of incubation at 4 C, three times; 1xTE, 4-5 min of incubation at room temperature, once.After washing, any remained liquid was removed with fine syringe needles.100 mL elution buffer (1xTE, 1% (w/v) SDS) was added, followed by incubation for 15 min at 65 C with 1,300 rpm on Eppendorf Thermomixer C. The sample was then spun down        (B) Heatmap showing the average interface predicted template modeling (ipTM) score of all five predicted models between S. pombe FACT subunits and each core replisome component.The ipTM and heatmap scale ranges from 0.25 to 0.7.Although the ipTM score for the Spt16 and Pri1 interaction is high, the interaction interface appears to be small and clashes with the interaction interface between Pri1 and Pri2 in the published cryo-EM primase structure (PDB: 8B9C). 96C) In vitro GST pull-down assays using the indicated GST-Mrc1 segments to pull-down endogenously purified FACT.SDS-PAGE gels show purified FACT (left), input (middle), and bound fractions (right).

Figure 1 .
Figure 1.The full fork protection complex is required for heterochromatin maintenance (A) Diagram showing the inducible ectopic heterochromatin system.(B) Diagram highlighting the location of the fork protection complex subunits (Swi1, Swi3, and Mrc1) on the replisome.(C)Heterochromatin maintenance assay testing the roles of subunits of the fork protection complex in epigenetic inheritance.10-fold serial dilutions of cells were plated on the indicated growth medium to detect heterochromatin establishment (ÀAHT) and maintenance (+AHT).Loss of growth on medium containing hydroxyurea (HU+) indicates deficiency in replication checkpoint.* denotes a stop codon.(D) H3K9me2 ChIP-qPCR at the 10XtetO-ade6 + locus showing that the H3K9me2 levels in mrc1 + or mrc1D cells at the establishment phase (ÀAHT) and the maintenance phase 24 h after growth in the presence of AHT.Error bars indicate standard deviations of 3 biological replicates.(E) Diagram illustrating the gene-targeted random mutagenesis of mrc1 + to isolate mutant cells that are competent for heterochromatin establishment and replication checkpoint but fail to maintain heterochromatin.(F) Separation-of-function alleles isolated from the random mutagenesis of mrc1 + that abolishes heterochromatin maintenance but not replication checkpoint.(G) IP-MS analysis of TAP-tagged heterochromatin maintenance-competent Mrc1-SSAA and mutant Mrc1-(1-620).(H) IP-MS of TAP-Sld5 in mrc1 + and mrc1-W620STOP cells.(G and H) x axis, the log 2 fold change between wild type and mutant epitope tagged proteins; y axis, normalized intensity of proteins associated with the indicated tagged proteins detected by mass spectrometry.See also FigureS1.

Figure
Figure 4. Mrc1 histone-binding activity is required for heterochromatin maintenance in S. pombe

4 .
Figure 4. Mrc1 histone-binding activity is required for heterochromatin maintenance in S. pombe (A) Energy minimized AlphaFold-predicted interaction between Mrc1-a2 and histone H4s.Top, diagram showing the location of Mrc1-a2 and the Mrc1-histonebinding domain.Bottom, hydrophobic map of the Mrc1-a2 and detailed predicted interactions between Mrc1-a2 and histone H4. (B) In vitro GST pull-down assays showing the effect of hydrophobic (Mrc1-M755A, F758A, L774A) and electrostatic (Mrc1-E763R, D767K) mutations in Mrc1-a2 on histone H3-H4 binding.(C) Heterochromatin maintenance assay showing the phenotypes of hydrophobic and electrostatic mutations in mrc1-a2.(D) Top, diagram showing the ade6 + reporter gene inserted at the boundary of the mating type locus IR-L (L(BglII)::ade6 + ).Bottom, silencing assays showing phenotypes of cells carrying Mrc1-histone-binding domain mutations in silencing of the ade6 + reporter.(E) Top, diagram showing the DNA sequence-dependent heterochromatin maintenance reporter system in S. pombe.Bottom, spotting assay showing the maintenance phenotype of the ura4 + report gene in wild-type cells and cells carrying the indicated mutations.As a control, cells with deletions of Atf1/Pcr1 binding sites (s1D,s2D) are unable to maintain heterochromatin.(F) H3K9me2 ChIP-qPCR analysis of mrc1 mutations in combination of ago1D at pericentromere dg repeats.Error bars indicate standard deviations of 3 biological replicates.See also Figure S4.

Figure 5 .
Figure 5.The histone-binding domain of Mrc1 promotes parental histone transfer without affecting transfer symmetry (A) Diagram illustrating the dual gene silencing reporter systems in S. cerevisiae.(B) Diagram of the predicted histone-binding domain and Mcm2/Cdc45 interaction region, PDB: 8B9C 96 and AlphaFold prediction (more details are presented in Figures S7J-S7P), in the Mrc1-like domain of S. cerevisiae Mrc1.(C) Growth assays showing the effects of the indicated mutations on silencing and replication stress.(D) eSPAN bias of the parental histone surrogate H3K4me3 (left) and the new histone surrogate H3K56ac (right) distribution around 139 early replicating origins (ACSs) in wild-type (WT), mrc1D, mrc1-like domainD, dpb3D, dpb3D mrc1D, and dpb3D mrc1-like domainD S. cerevisiae cells.(E) eSPAN bias of parental histone surrogate H3K4me3 (left) and the new histone surrogate H3K56ac (right) around 139 early ACSs in wild-type (WT), mrc1D, mcm2-3A, and mrc1D mcm2-3A S. cerevisiae cells.(F) eSPAN bias of the parental histone H3K4me3 distribution in MRC1, mrc1-a2D S. cerevisiae cells.(G)eSPAN bias of parental histone surrogate H3K4me3 distribution around 162 origin of replication in wild-type (WT), mrc1-3A, mcm2-2A S. pombe cells.The shading of the bias line plot is the 95% confidence interval of mean value of at least two biological replicates, which is mean ± 2 folds of the standard error.See also Fang et al.97 for WT and mcm2-2A eSPAN analysis.(H) Violin plot showing the average of two biological replicates of S. pombe eSPAN H3K4me3 density on the leading and lagging strand around the replication origin (2.5 kb upstream of replication origin to 2.5 kb downstream of replication origin).The numbers in the figure represent changes of eSPAN density over wildtype cells for each strand.*** indicates p value < 0.001 (two-sample t test).(I) Diagram illustrating a parental H3K9me2 maintenance assay.Top, diagram of the S. pombe reporter system that lacks read-write activity.Bottom, diagram of the designed assay to analyze the maintenance of H3K9me2 in a synchronized cell population after 6 h after release from cell cycle arrest.(J) ChIP-qPCR of parental H3K9me2 in wild-type (WT), mcm2-3A, mrc1-3A cells 6 h after release from cell cycle arrest.A two-tailed two-sample t test with unequal variance was used for statistical significant test between wild-type and mutant samples.Error bars indicate standard deviation of 5 biological repicates.* p value < 0.05, ** p value < 0.01, n.s., not significant (p = 0.068).See also FigureS5.
(H) Violin plot showing the average of two biological replicates of S. pombe eSPAN H3K4me3 density on the leading and lagging strand around the replication origin (2.5 kb upstream of replication origin to 2.5 kb downstream of replication origin).The numbers in the figure represent changes of eSPAN density over wildtype cells for each strand.*** indicates p value < 0.001 (two-sample t test).(I) Diagram illustrating a parental H3K9me2 maintenance assay.Top, diagram of the S. pombe reporter system that lacks read-write activity.Bottom, diagram of the designed assay to analyze the maintenance of H3K9me2 in a synchronized cell population after 6 h after release from cell cycle arrest.(J) ChIP-qPCR of parental H3K9me2 in wild-type (WT), mcm2-3A, mrc1-3A cells 6 h after release from cell cycle arrest.A two-tailed two-sample t test with unequal variance was used for statistical significant test between wild-type and mutant samples.Error bars indicate standard deviation of 5 biological repicates.* p value < 0.05, ** p value < 0.01, n.s., not significant (p = 0.068).See also Figure S5.

Figure 6 .
Figure 6.Identification of FACT binding sites in the replisome and their requirement for heterochromatin maintenance (A) Predicted structure of Swi1 and FACT subunit Spt16.(B) The predicted interacting domains of Spt16 and Swi1 in (A) are highlighted in yellow and orange, respectively.(C) Heterochromatin maintenance assay showing the effects of swi1, mrc1, and mcm2 mutations.(D) Diagram of regions in the N-terminal extension (NTE) of Pol1 predicted to interact with Spt16, (H3.1-H4) 2 , and the Mcl1 C-terminal domain (CTD).The predicted interacting domains of Spt16 and Mcl1 in (F) are highlighted in green and yellow, respectively.(E) Predicted structure of Pol1-NTE (a1, a2, and a3) with Spt16-middle domain (MD), (H3.1-H4) 2 and Mcl1-CTD.(F) Heterochromatin maintenance assay showing the effect of the indicated pol1 mutations.(G-I) In vitro GST pull-down assays showing the interaction of the indicated GST-Pol1-NTE proteins with purified FACT complex (G), (H3-H4) 2 (H), and Mcl1-(CTD) (I).See also Figures S6 and S7. .

Figure 7 .
Figure 7. Mrc1 acts as a parental histone distribution site (A) The predicted location of Mrc1-(H3-H4) 2 on the cryo-EM structure of the replisome (PDB: 8B9C and 7QHS).Top, diagram showing the indicated regions in Mrc1 involved in interaction with multiple replisome components, replication checkpoint signaling, and interaction with histones.The predicted Pol2 (Pol ε)interacting region was identified by AlphaFold-Multimer and is consistent with previous biochemical results.102The newly identified histone-binding region is highlighted in pink and the Cdc45/Mcm2(NTD) interacting region is highlighted in red.Bottom, the predicted structure of Mrc1-like domain/(H3-H4) 2 /Cdc45/ Mcm2(NTD) was aligned to the cryo-EM structure (PDB: 8B9C) via the Mrc1-like domain a5 helix.See Figures S7K-S7P for alignment details. (B) Model for DNA replication-coupled directional parental histone transfer with FACT acting as a mobile chaperone.P, parental site; D, distribution site; LD1, leading strand site 1; LG1 and LG2, lagging strand sites.See text for details.See also FigureS8.

Figure S3 .
Figure S3.Structural analysis of histone-binding activities of replisome components predicted by AlphaFold-Multimer, related to Figure2

(
A) Top, diagram illustrating the location of Mcm2 histone-binding domain (HBD) at the N-terminal extension of Mcm2.Bottom, predicted structures of S. pombeMcm2-HBD with H3.1-H4 tetramer, modified crystal structure of human MCM2-HBD with H3.3-H4 tetramer (PDB: 5BNV),33 and alignment of the two structures.Conserved amino acids involved in histone binding and heterochromatin maintenance (FigureS2B) are highlighted in the structure.(B) Top, diagram illustrates predicted Spt16 histone interaction domains.Bottom, predicted structure of Spt16-middle domain and C-terminal domain (MD/CTD) interacting with H3.1-H4 tetramer, published crystal structure of human SPT16-(MD/CTD) with H3.1-H4 tetramer (PDB: 4Z2M)46 and alignment of the two structures.(C) Top, diagram illustrates the regions at the N-terminal extension (NTE) of Pol1 predicted by AlphaFold.The a2 helix, predicted to bind to histone H3-H4, is highlight in magenta color.Middle, the predicted structure of Pol1(NTE)-H3.1-H4.The amino acids that are conserved and required for heterochromatin maintenance (FigureS2B) are highlighted in the model.Bottom, the PAE plot of the predicted structure of S. pombe Pol1(NTE) with the H3-H4 tetramer.(D) Top, the domains in S. pombe histone-like proteins Dpb3 and Dpb4 predicted by AlphaFold-Multimer.Middle, the predicted structure of S. pombe Dpb3-Dpb4-H3.1-H4tetramer.Bottom, the PAE plot of the predicted structure of Dpb3-Dpb4-H3-H4 tetramer.(E) Top, the domains in human MCM10 predicted by AlphaFold-Multimer.Middle, predicted structure of human MCM10-H3.1-H4tetramer.Bottom, the PAE plot of the predicted structure of human MCM10-H3.1-H4tetramer.(F) Left, the crystal structure of nucleosome core particle (PDB: 1AOI)26 used for alignment.Right, the predicted structure of Mrc1-like domain-(H3.1-H4) 2 used for alignment.(G) Alignment of predicted structure of Mrc1-like domain with (H3.1-H4) 2 and the crystal structure of nucleosome core particle shows the locations of a1-3 of Mrc1-like domain in the predicted structure relative to the location of nucleosomal DNA (from Dyad to SHL-3), histone H2B-a2 and histone H2A-C-terminal extension (CTE), respectively.The a2-3 of Mrc1-like domain tilts $10.8 , compared with H2B-a2 and H2A-CTE.For illustration purposes, only the relevant regions of nucleosomal DNA and histones are shown.

Figure S4 .
Figure S4.Interactions between Mrc1-like domain and histones, related to Figure 3

(
A) PAE plots of predicted interaction between S. pombe Mrc1-like domain and H2A-H2B dimer.(B) GST-Mrc1-like domain protein from S. pombe binds H2A-H2B weakly under stringent binding conditions (500 mM NaCl).(C) GST-Mrc1-like domain fusion proteins from S. pombe and human pull-down histone H3-H4 under stringent binding conditions (500 mM NaCl).(D) GST pull-down assays showing that the interaction between S. cerevisiae Mrc1-like domain and H3-H4 is salt-sensitive.(E) SEC-MALS distribution of Mrc1-(651-900)-(H3-H4) 2 complex.Black curve indicates cumulative molar mass in the range of the indicated molar mass, and red curve indicates linear differential molar mass at the indicated molar mass.(F) Diagram summarizing mutations of the Mrc1-like domain that specifically abolish heterochromatin maintenance isolated from targeted mutagenesis or generated based on structural predictions.(G) Bar plot showing the percentage of red or variegated cells that maintain heterochromatin in the indicated mrc1 mutant cells in (E); mrc1-3A (mrc1-M755A,F758A,L774A).n = 3. Error bars indicate the standard deviation.

Figure S6 .
Figure S6.Structural predictions suggest interactions between FACT and replisome components, related to Figure6

(
A) IP-MS analysis of TAP-tagged Sld5 from mrc1 + and mrc1-3A cells.Colors indicate replisome components shown below the plot.
(D) PAE of the AlphaFold-Multimer predicted interaction between Spt16-Mrc1.(E) Based on the GST pull-down results, the first interaction between Mrc1 and FACT localizes at the N terminus of Mrc1 (FBD1, amino acids 134-168) and the Spt16-middle domain.The location of other binding domains is shown for reference.(F) The predicted structure of Mrc1-FBD1 in complex with Spt16-DD-MD and H3-H4 tetramer.(G) The PAE plot of the predicted structure in (F).(H) The second interaction interface between Spt16 and Mrc1 localizes to a middle region of Mrc1 (Mrc1-FBD2, amino acids 513-540) and the N-terminal domain of Spt16 (Spt16-NTD).(I) The predicted structure of Spt16 (without CTD)-Mrc1(middle region including FBD2)-H3-H4 tetramer.(J) The PAE plot of the predicted structure in (I).

Figure S8 .
Figure S8.Structural predictions suggest that the Mrc1 histone-binding domain can bind H3-H4 tetramer together with other replisome histone-binding domains, related to Figure 7

TABLE
Please cite this article in press as:Yu et al., A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance, Cell (2024), https://doi.org/10.1016/j.cell.2024.07.006Please cite this article in press as: Yu et al., A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance, Cell (2024), https://doi.org/10.1016/j.cell.2024.07.006 ll e1 Cell 187, 1-19.e1-e9,September 5, 2024 (Continued on next page) ll Cell 187, 1-19.e1-e9,September 5, 2024 e2 the project name PRJCA018248) and S. pombe eSPAN mrc1-3A data are deposited at Genome Expression Omnibus (GSE269383) and are publicly available on the date of publication.Accession numbers for all datasets are listed in the key resources table.d The codes used to generate and analyze the datasets were deposited at Mendeley Data at https://doi.org/10.17632/jhzmfr8bbs.1 and are publicly available on the date of publication.d Any additional information that is required for reanalyzing the data reported in this study is available from the lead contact upon request.
d The raw gel, membrane, silencing assay images were deposited at Mendeley Data at https://doi.org/10.17632/jhzmfr8bbs.1 and are publicly available on the date of publication.All AlphaFold-Multimer-predicted structures and modeled structures are deposited on ModelArchive under the accession number ma-dm-hisrep and are publicly available on the date of publication.S. cerevisiae eSPAN data are deposited at Genome Research Archive (accession number CRA011810 and CRA014983 under Identification and alignment of Mrc1-like domain among eukaryotic species Mrc1-like domain is annotated among fungi as the PF09444 in the Pfam database.Additional Mrc1-like domains among other eukaryotic species were identified by aligning fission yeast Mrc1-like domain with full-length Mrc1/CLASPIN homologs in each species Size-exclusion chromatography with multi-angle light scattering Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments were performed with the SEC-MALS system at Harvard Medical School CMI core facility.The SEC-MALS contains an Agilent 1260 Infinity LC System with variable UV detector connected with a Superdex 200 increase 3.2/100 column (Cytiva), a Wyatt Dawn Heleos II MALS detector, and a Wyatt Optilab T-rEX Refractive Index Detector.The SEC column was equilibrated with SEC-MALS buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.5 mM TCEP) overnight at 25 C. First, 80 mL 30 mM monodispersed BSA (Thermo Scientific) was spun at 14,000 rpm for 10 min and injected into the SEC-MALS system at the flow of 0.045 mL/min at 25 C through the Agilent autosampler.Peak alignment, band broadening, light scattering detector normalization were performed on the monodispersed BSA monomer peak.Then 80 mL 25-50 mM Mrc1-like domain, H3-H4 tetramer, or Mrc1-like domain-H3-H4 tetramer complex samples were applied to SEC-MALS using the same conditions as the BSA sample.Data were analyzed under the BSA control setting and visualized using ASTRA (version 7.3.2.21).CodonPlus competent cells were transformed with pET28a vectors expressing the fusion of 6xHis-SUMO and S. pombe Mcl1-CTD domain.pET28a vector containing BL21-CodonPlus competent cells were cultured in 1 L LB media with 50 mg/mL ampicillin and 25 mg/mL chloramphenicol and induced with 2% ethanol and 0.2 mM IPTG at 20 C for 4 hours with shaking at 220 rpm starting with OD 600 =0.7$0.9.Cells were collected by centrifugation at 7,000 rpm for 20 min at 4 C. Cell lysate were generated as described above with the addition of 20 mM imidazole and in the absence of EDTA.Clear lysate was incubated with 1 mL chelating resin at 4 C for 30 min with rotation.The resin was put on a chromatography column and washed with Wash/Equilibrium buffer (40 mM imidazole) five times.The resin was then equilibrated in the elution buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 400 mM imidazole, 2 mM b-mercaptoethanol, 10% (v/v) glycerol).Ulp1 protease was added into the elution buffer to cleave the Mcl1-CTD domain from the 6xHis-SUMO in a dialysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM imidazole, 2 mM b-mercaptoethanol, 10% (v/v) glycerol) at 4 C overnight.Supernatant containing the eluted protein was subjected to chelating resin once to remove 6xHis-SUMO.Sample was then subjected to HiTrap Q HP 1 mL (Cytiva) with a 20 CV gradient of NaCl from 100 mM to 1 M. Peak fractions containing Mcl1-CTD domain was eluted around 220 mM NaCl and concentrated using Amicon 10 MWCO Ultra-4 Centrifugal Filter Unit.The protein was then further purified Superdex 200 increase 10/300 GL in 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM DTT, 10% (v/v) glycerol.